CDOM Absorption Measurements

Water samples for measurement of absorption by coloured dissolved organic material (CDOM) should be taken directly from the carboy (collected using the hand-held Niskin bottle or the flow-through system – see “Overall sampling procedure”).

Sample Preparation

Note: Filtering should be done immediately after sample collection.

1) Soak 47mm 0.2m Nucleopore membrane filters in 10% HCl solution for 15 minutes and then rinse thoroughly with Super Q-water. 2) Place a filter on the filter support. Small wrinkles don’t matter, but make sure you cover the entire frit and make sure you have a good seal between the filter funnel and the filter support. 3) Filter about 30-50ml of Q-water through the filter. Swirl in filter flask, and discard this water. This Q-water rinse and the sample rinses in the next step get rid of any leachate that might initially come from the filter. 4) Pour about 10ml of sample into the filter funnel and filter this through. Swirl in filter flask and discard water. Repeat this twice more. The purpose here is to get rid of any leachate that might initially come from the filter and to make sure all Q-water droplets are out of the filter flask to avoid dilution of the filtered sample. 5) Filter approx. 200 ml of sample, which should provide enough filtered water for rinsing of the cuvette and to run two absorption spectra (if necessary). 6) Make a note in the logbook anytime you need to use more than one filter for a sample bottle. 7) Rinse the beaker you used to pour the sample into the filtration apparatus with Q- water three times. The objective here is to get rid of anything from the unfiltered sample that may have stuck to the sides of the beaker. 8) Rinse the beaker 3 times with the filtered sample from the filter flask. The objective here is to remove any remaining Q-water droplets in the bottle to avoid diluting the sample. 9) Fill the beaker with the remaining filtered sample in the filter flask. Cover with aluminum foil. 10) If you are going to scan the samples immediately after filtering the batch, then set the beaker, covered with aluminum foil to protect from the light, out on the counter to warm to room temperature. If running at a later time/date, put the covered beaker back in the refrigerator and store at 4°C until ready to scan. 11) Discard the filter – Do NOT use the same filter for more than one sample bottle. They almost always clog partway through the second sample bottle.

Spectrophotometric Measurement Procedure

Scanning of the samples should be done within 24 hours of sampling.

IMPORTANT: Remove the mylar sheet from the spectrophotometer before any CDOM scans are conducted, otherwise your readings in the UV will be very erratic.

1) Turn on the Cary spec by turning on the computer. Goto the Start > Programs > CARY WINUV > Scan. Warm up the spec for 30 minutes. 2) Open Scan method file (File > Open Method – goto C:\JAMSTEC folder, CDOM sub-folder and open CDOM_method.MSW). The parameters should be correctly specified, but you can check the setup yourself (page 4-16 manual). For CDOM we scan the samples between 250 and 750 nm, Y axis 0-1, scan speed medium, baseline correction, enter your name in the reports tab, files will be converted into comma- delimited (csv) format. 3) Make sure the sampling compartment of the spec is clean and as dust-free as possible. The compartment may be cleaned with ethanol and kimwipes ONLY. 4) Run an air baseline by clicking on the BASELINE toggle. Now click on the “Traffic Lights” to record the air baseline. The spectrum should be visibly flat at zero, with noise less than  0.005. 5) The cuvette should have been stored with Q-water in them. Empty and rinse the cuvette a few times with fresh Q-water. Fill the cuvette with Q-water and make sure to rinse the stoppers in Q-water before stopping the cuvette. 6) When filling cuvette with Q-water, use a pre-cleaned glass beaker (used only for Q- water reference!). Try not to generate a lot of bubbles by slowly pouring the Q-water into the cuvette since bubbles tend to aggregate and move during a scan. It’s difficult to avoid bubbles altogether. If you do end up with some little bubbles, after you have put the stoppers in the cuvette, turn the cuvette so one stopper is higher than the other and tap the cuvette lightly (with a kimwipe covering the end of your finger as you tap – of course). 7) Wipe off any water on outside of cuvette with kimwipes. 8) Thoroughly clean the outside of cuvette with ethanol and kimwipes. Check the outside of the cuvette to make sure it is truly clean (no lint! no fingerprints!) and place it in the cuvette holder. Make sure 6Q lettering on the stopper neck is facing forward and to the right. Gently slide the cuvette to the left (Detector) side of the sample holder as far as it will go to ensure that the cuvette is in the identical position for each scan. 9) Run the Q-water sample as a sample by clicking on the “Traffic Lights” and record the spectrum. 10) Compare the pure water spectra with the pre-existing pure water spectra (filename C:\JAMSTEC\CDOM\milliq1.csv). They should be nearly identical in shape and magnitude. 11) Once you are satisfied that the Q-water reference sample, record the Q-water as a baseline by clicking on the BASELINE toggle. 12) Now run the Q-water reference as a sample by clicking on the ‘Traffic Lights’. The resulting spectrum should be zero with relatively little scatter (between  0.005). 13) Remove the cuvette from its holder, empty it, swirl the sample beaker to make sure it’s well mixed (mainly for uniform temperature at this point), rinse the same cuvette 3 times with about 5-10ml sample, fill the cuvette with sample, clean the outside of the cuvette with ethanol and kimwipes and replace it in the holder. Close the sampling compartment and scan by clicking on the ‘Traffic Lights’. 14) Before starting the next sample rinse the sample cuvette 4 times with Q-water and refilling it, making sure to rinse the stoppers in Q-water as well. 15) When the last scan is finished, turn off the spec. Leave the cuvettes filled with Q- water when storaging. 16) Acid rinse the beakers and prepare them for the next set of samples. Cover with aluminum foil.

NOTE ON CUVETTE HANDLING: Avoid touching the cuvette with your bare fingers, especially at the ends. Fingerprints and oils in the skin have optical properties that will show up in the scans if you are not careful about keeping the cuvettes clean. The same goes for glove prints if you use gloves. Ideally, only kimwipes actually touch the cuvettes.

Filtered samples need to be at room temperature before scanning to avoid condensation on the cuvettes. Additionally, you do not want samples to be sitting out for extended periods of time and samples can degrade with exposure to light, so do not set sample bottles out too far in advance and try to keep them as dark as possible.

Problems and Situations

 There isn’t enough water in the sample bottle to do 5-10ml rinses in triplicate for each scan. The cuvette needs to be rinsed at least once before filling it for the first replicate to remove any remaining Q-water from the cuvette; 2-3 rinses is better. Rinses can be eliminated if absolutely necessary before the second and third replicate. Rinses before the second and third replicate are done to loosen and remove anything that might be sticky and accumulate on the inside of the cuvette when refilling with additional sample.  There is oil (or something else extremely sticky) in the sample. Rinse the inside of the cuvette with ethanol until it runs clear (the ethanol will be cloudy if there is a lot of oil in the cuvette). Rinse the cuvette 3 times with Q-water to make sure the ethanol is removed from the cuvette, then continue rinsing and filling the cuvette with sample (3 rinses and fill) or Q-water (6 rinses and fill) as normal. Do the ethanol rinse between each scan/replicate. For most samples, one or two ethanol rinses are all that is needed.  A dip appears in the spectrum around 715nm and a peak around 740-750nm. These are temperature/salinity dependent and cannot be avoided. Wavelengths greater than 700nm are eliminated in post-processing due to this problem. Since you can’t do anything to remove this feature from the spectrum, just ignore it.  The spectrum is highly erratic or has unexpected "features." Smooth bumps covering a range greater than 10nm can be real features of sample absorption, but jagged or highly variable spectra are usually a "feature" originating from a dirty cuvette. Whenever you have doubt as to whether a spectrum is correct, first check the cuvettes to make sure they are clean. If a piece of lint/dust/dirt gets on the reference cuvette, absorption values will go erratically negative. Lint/dust/dirt on the sample cuvette will cause absorption values to increase. The middle of the ends of the cuvettes are the most critical areas that must remain free of all lint, dust, dirt, fingerprints, gloveprints, scratches, condensation, and residues of any sorts. Rescan the sample.

Queries: Contact Heather Boumann Email: [email protected]